Ca2+ allostery in PTH-receptor signaling

Abstract

Significance Blood Ca2+ homeostasis is maintained by the actions of the parathyroid hormone (PTH) on its cognate receptor PTHR. PTH binding to PTHR mediates prolonged adenosine 3′,5′-cyclic monophosphate (cAMP) responses in cells after receptor internalization. Here, we show that extracellular Ca2+ prolongs the residence time of ligands on the receptor, consequently, increasing both the duration of receptor activation and cAMP signaling. This positive Ca2+ allostery is lost for the PTH mutant R25C, recently identified as a new cause of hypocalcemia in humans. Using mass spectrometry approaches, we identify acidic clusters within the first extracellular loop of PTHR as determinants of Ca2+ allostery and endosomal cAMP signaling. These findings provide insight into the molecular etiology of hypocalcemia and disease relevance of endosomal cAMP signaling. The parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only the PTH sustains GS-mediated adenosine 3′,5′-cyclic monophosphate (cAMP) production after PTHR internalization into early endosomes. The mechanism of this unexpected behavior for a G-protein–coupled receptor is not fully understood. Here, we show that extracellular Ca2+ acts as a positive allosteric modulator of PTHR signaling that regulates sustained cAMP production. Equilibrium and kinetic studies of ligand-binding and receptor activation reveal that Ca2+ prolongs the residence time of ligands on the receptor, thus, increasing both the duration of the receptor activation and the cAMP signaling. We further find that Ca2+ allostery in the PTHR is strongly affected by the point mutation recently identified in the PTH (PTHR25C) as a new cause of hypocalcemia in humans. Using high-resolution and mass accuracy mass spectrometry approaches, we identified acidic clusters in the receptor’s first extracellular loop as key determinants for Ca2+ allosterism and endosomal cAMP signaling. These findings coupled to defective Ca2+ allostery and cAMP signaling in the PTHR by hypocalcemia-causing PTHR25C suggest that Ca2+ allostery in PTHR signaling may be involved in primary signaling processes regulating calcium homeostasis.